Dry eye disease has been estimated to affect from 5% through 30% of the population depending on age and diagnostic criteria. Common tests for dry eye are subjective (e.g. questionnaires) or invasive (e.g. Schirmir Test, Tear Breakup Time, Vital Staining). More complex technologies are not simple or rapid enough to be useful as a screening test (protein levels, interferometry). In general, the repeatability of common tests for dry eye is not very good. A 2004 study of dry eye patients concluded the repeatability of objective slit lamp tests was poor and Schirmir test readings “fluctuate severely” from visit to visit. A 2008 review describes problems with variability and reproducibility of common dry eye disease diagnostic tests.
The International Dry Eye Workshop (2007) defined dry eye as “a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.”
The present invention specifically addresses the “increased osmolarity” described in the definition. The International Dry Eye Workshop (2007) recognized hyperosmolarity as an attractive “signature feature” of dry eye disease. Tomlinson et al. describe tear osmolarity as “[a] single biophysical measurement that captures the balance of inputs and outputs from the tear film dynamics.” A. Tomlinson et al., “Tear Film Osmolarity: Determination of a Referent for Dry Eye Diagnosis,” Investigative Ophthalmology and Visual Science, vol. 47, pages 4309-4315 (2006). The first studies of individual tear fluid osmolarity were reported in 1978. These reports used freezing point and dew point depression to determine osmolarity and found significant differences between normal and dry eye disease patients. Numerous studies since 1978 have confirmed osmolarity is a key parameter in the diagnosis of dry eye disease. Osmolarity has been discussed as the new “Gold Standard” in diagnosing dry eye disease as early as 1986 and as recently as 2007.
Objective osmolarity tests of tear fluid are possible, however they have seen limited use in the diagnosis of dry eye due to difficulties in making the measurements on sub-microliter samples, the high level of technical competency required and the limited availability of instruments to health care practitioners. Technologies used for determining tear osmolarity include freezing point depression, vapor pressure (dew point) and conductivity. A common problem with these tests is the very broad and overlapping normal distributions seen in comparing normal and dry eye subjects. Typical averages and standard deviations observed in osmolarity tests as summarized by Tomlinson et al. are shown in Table 1.
TABLE 1NormalDry EyeTestSubjectsSubjectsFreezing Point304.4 +/− 7.2329.6 +/− 17  DepressionConductivity296.4 +/− 30 324 +/− 41Average from  302 +/− 9.7326.9 +/− 22.116 references
Preliminary data is also available on a newer conductivity based “lab on a chip” instrument from Tearlab Corporation (San Diego, Calif.). The Food and Drug Administration (FDA) 510(k) Performance Testing Summary for this instrument shows a normal range of 294+/−5.5 and moderate dry eye range of 316+/−4.5. This data was generated using “contrived tear” samples with constant salts, protein and lipid. Additional variation was seen between instruments, sites and within and between lot variation in the disposable lab-on-a-chip collection/electrode device.
U.S. Pat. No. 7,395,103 B2 describes a surface plasmon resonance (SPR) device for determining tear osmolarity. SPR-based sensors use a gold surface to contact and read the refractive index of the sample. No data is currently available for this instrument. It is well known that proteins quickly adsorb to metallic surfaces used for SPR. The signal from this device would thus be a combination of the bulk (solution) refractive index and the adsorbing protein.
Measuring the refractive index of tear fluid by critical angle refractometry alone has been previously proposed and tested as a method to measure tear osmolarity See J. P. Craig et al., “Refractive Index and Osmolality of Human Tears,” Optometry and Vision Science, vol. 72, pages 718-724 (1995). The correlation between refractive index and osmolarity is well established for salt solutions. Craig et al. found no correlation between refractive index and osmolarity measured by freezing point depression and a slight correlation between lactoferrin concentration and refractive index. No attempts were made to correct the refractive index reading for changing protein concentration.
The relatively high variability observed when measuring tear osmolarity is likely due to a number of factors including those inherent in the measuring technology and those contributed by the sample. Instruments which measure freezing point depression and vapor pressure require very accurate temperature measurements in an environment where temperature is changing rapidly. Conductivity measurements require the sample be brought into contact with electrodes and an electrical current passed through the sample. Conductivity measurements are also both temperature and volume dependent.
It is highly likely that sample composition plays an important role in the variability of tear fluid measurements. Tears are complex mixtures of many solutes, the vast majority of which are salts (NaCl; KCl) and proteins. Dissolved salts and proteins contribute approximately equally to the total solute mass of the tear. Osmolarity is a function of the molar concentration or number of molecules of a solute per volume of solution. Because of their large mass and relatively low molarity, proteins have a greatly diminished effect on tear osmolarity relative to salts, which have higher molar concentrations in tear fluid; at equal mass, the effect of proteins on osmolarity is on the order of 1,000 fold less than the effect of salts. The effect of samples with varying protein concentrations on both conductivity and freezing point/vapor pressure measurements is not well understood and is a likely candidate for a part of the observed variability. A 2004 study concluded significant errors were introduced when protein concentration was varied during conductivity based hemoglobin and hematocrit assays. Proteins also have a tendency to coat and bind to metallic surfaces fairly quickly. Electrode “fouling” is a common occurrence when measuring the conductivity of protein containing solutions. Measurement errors are the result of a high resistance and/or electrochemical reactions on coated metallic electrodes.
Thus, there is a need for an apparatus and a method capable of accurately and precisely measuring tear fluid osmolarity. The apparatus and method should be easy to use with respect to sub-microliter tear fluid samples.